The increasing incidence of antibiotic resistance hampers our ability to effectively treat bacterial infections. The situation is especially dire for bacteria like Pseudomonas aeruginosa, which have limited treatment options. Cationic antimicrobial peptides (CAPs) are currently used as last resort treatments for multidrug resistant P. aeruginosa. Alarmingly, CAP-resistant strains have been encountered. To extend the utility of CAPs, the research project proposes to develop small molecule inhibitors of the resistance pathway in P. aeruginosa that can be used in a combination therapy along with a CAP. P. aeruginosa becomes resistant to CAPs by modifying the lipids of its outer membrane with carbohydrates, decreasing the overall negative charge of the membrane, and preventing the association and cell entry of the positively-charged CAP. The research proposes to inhibit ArnA, the first committed enzyme of the modification pathway. We will expand upon our initial ArnA inhibitors using a focused library of compounds structurally related to the initial hit. The impact of the structure of the small molecules on their ability to inhibit ArnA will then be analyzed (Aim 1). Inhibition of ArnA should prevent the remodeling of the outer membrane, forcing P. aeruginosa to maintain a negatively-charged outer membrane. The charge state of the membrane after inhibitor treatment will be characterized, and modifications to lipid A will be directly analyzed by mass spectrometry (Aim 2). The dissociation constant (Kd) of top inhibitors and the type of inhibition exhibited will be identified in a set of binding and kinetics experiment (Aim 3). Results from Aims 1 and 3 will guide the design and synthesis of a second set of inhibitors that aim to have even better potency and pharmacological properties. The long-term goal of the project is to develop a combination therapy that can be used to treat not only drug-resistant P. aeruginosa, but other gram-negative pathogens of concern like carbapenem-resistant Enterobacteriaceae.